Sickle cell disease has been recognized within West Africa for several centuries. Sickle cell anemia and the existence of sickle hemoglobin (Hb S) was the first genetic disease to be understood at the molecular level. It is recognized today as the morphological and clinical result of a glycine to valine substitution at the No. 6 position of the beta globin chain (Ingram, Nature 178: 792-794 (1956)). The origin of the amino acid change and of the disease state is the consequence of a single nucleotide substitution (Marotta et al., J. Biol. Chem. 252:5040-5053 (1977)).
The major source of morbidity and mortality of patients suffering from sickle cell disease is vascular occlusion caused by the sickled cells, which causes repeated episodes of pain in both acute and chronic form and also causes ongoing organ damage with the passage of time. It has long been recognized and accepted that the deformation and distortion of sickle cell erythrocytes upon complete deoxygenation is caused by polymerization and intracellular gelation of sickle hemoglobin, hemoglobin S (Hb S). The phenomenon is well reviewed and discussed by Eaton et al., Blood 70:1245 (1987). The intracellular gelatin and polymerization of Hb S can occur at any time during an erythrocyte's journey through the vasculature. Thus, erythrocytes in patients with sickle cell disease containing no polymerized hemoglobin S may pass through the microcirculation and return to the lungs without sickling, sickle in the veins, or sickle in the capillaries.
The probability of each of these events is determined by the delay time for intracellular gelation relative to the appropriate capillary transit time (Eaton, et al., Blood 47: 621(1976)). In turn, the delay time is dependent upon the oxygenation state of the hemoglobin, with deoxygenation shortening the delay time. If it is thermodynamically impossible for intracellular gelation to take place, or if the delay time at venous oxygen pressures is longer than about 15 seconds, cell sickling will not occur. If the delay time is between about 1 and 15 seconds, the red cell will likely sickle in the veins. If the delay time is less than about 1 second, red cells will sickle within the capillaries.
For red cells that sickle within the capillaries, a number of consequent events are possible. These range from no effect on transit time, to transient occlusion of the capillary, to a more permanent blockage that may ultimately result in ischemia or infarction of the surrounding cells, and in the subsequent destruction of the red cell.
Normal erythrocytes are comprised of approximately 70% water. Water crosses a normal erythrocyte membrane in milliseconds. Loss of cell water causes an exponential increase in cytoplasmic viscosity as the mean cell hemoglobin concentration (MCHC) rises above about 32 g/dL. Since cytoplasmic viscosity is a major determinate of erythrocyte deformability and sickling, the dehydration of the erythrocyte has substantial rheological and pathological consequences. Regulation of erythrocyte dehydration is recognized as an important therapeutic approach for treating sickle cell disease. Since cell water follows any osmotic change in intracellular ion concentration, maintaining the red cell's potassium concentration is of particular importance (Stuart et al., Brit J Haematol. 69:1-4 (1988)).
Many approaches to therapeutically treating dehydrated sickle cells (thus decreasing polymerization of hemoglobin S by lowering the osmolality of plasma) have been tried with limited success, including the following approaches: intravenous infusion of distilled water (Gye et al., Am. J. Med. Sci. 266: 267-277(1973)); administration of the antidiuretic hormone vasopressin together with a high fluid intake and salt restriction (Rosa et al., M. Eng. J. Med. 303:1138-1143 (1980); Charache et al., Blood 58: 892-896 (1981)); the use of monensin to increase the cation content of the sickle cell (Clark et al., J. Clin. Invest. 70:1074-1080 (1982); Fahim et al., Life Sciences 29:1959-1966 (1981)); intravenous administration of cetiedil citrate (Benjamin et al., Blood 67: 1442-1447 (1986); Berkowitz et al., Am. J. Hematol. 17: 217-223 (1984); Stuart et al., J. Clin. Pathol. 40:1182-1186 (1987)); and the use of oxpentifylline (Stuart et al., supra).
Another approach towards therapeutically treating dehydrated sickle cells involves altering erythrocyte potassium flux by targeting a calcium-dependent potassium channel (Ishi et al., Proc. Natl. Acad. Sci. 94(21): 11651-11656 (1997)). This calcium activated potassium channel is also referred to as the Gardos channel (Brugnara et al, J. Clin. Invest. 92: 520-526 (1993)). Recently, a cloned human intermediate conductance calcium activated potassium channel, hIK1, was shown to be substantially similar to the Gardos channel in terms of both its biophysical and pharmacological properties (Ishi, supra).
Methods that have been used to inhibit the Gardos channel include the administration to erythrocytes of imidazole, nitroimidazole and triazole antimycotic agents such as clotrimazole (U.S. Pat. No. 5,273,992 to Brugnara et al.). Clotrimazole, an imidazole-containing antimycotic agent, has been shown to be a specific, potent inhibitor of the Gardos channel of normal and sickle erythrocytes, and prevents Ca2+-dependent dehydration of sickle cells both in vitro and in vivo (Brugnara, supra; De Franceschi et al., J. Clin. Invest. 93: 1670-1676 (1994)). When combined with a compound which stabilizes the oxyconformation of Hb S, clotrimazole induces an additive reduction in the clogging rate of a micropore filter and may attenuate the formation of irreversibly sickled cells (Stuart et al., J. Haematol. 86:820-823 (1994)). Other compounds that contain a heteroaryl imidazole-like moiety believed to be useful in reducing sickle erythrocyte dehydration via Gardos channel inhibition include miconazole, econazole, butoconazole, oxiconazole and sulconazole. Although these compounds have been demonstrated to be effective at reducing sickle cell dehydration, other imidazole compounds have been found incapable of inhibiting the Gardos channel and preventing loss of potassium.
Since sickle cell anemia is a chronic disease, agents designed for treating it will ideally exhibit certain characteristics that are less essential in drugs for treating resolvable illnesses (e.g., fungal infections). A clinically useful Gardos channel inhibitor will exhibit extremely low toxicity over a prolonged course of administration, will have an excellent bioavailability, will be highly specific for the Gardos channel and will be potent in its interactions with this channel.
As can be seen from the above discussion, reducing sickle erythrocyte dehydration via blockade of the Gardos channel is a powerful therapeutic approach towards the treatment and/or prevention of sickle cell disease. Compounds capable of inhibiting the Gardos channel as a means of reducing sickle cell dehydration are highly desirable, and are therefore an object of the present invention.
Cell proliferation is a normal part of mammalian existence, necessary for life itself. However, cell proliferation is not always desirable, and has recently been shown to be the root of many life-threatening diseases such as cancer, certain skin disorders, inflammatory diseases, fibrotic conditions and arteriosclerotic conditions.
Cell proliferation is critically dependent on the regulated movement of ions across various cellular compartments, and is associated with the synthesis of DNA. Binding of specific polypeptide growth factors to specific receptors in growth-arrested cells triggers an array of early ionic signals that are critical in the cascade of mitogenic events eventually leading to DNA synthesis (Rozengurt, Science 234:161-164 (1986)). These include (1) a rapid increase in cystolic Ca2+, mostly due to rapid release of Ca2+ from intracellular stores; (2) capacitative Ca2+ influx in response to opening of ligand-bound and hyperpolarization-sensitive Ca2+ channels in the plasma membrane that contribute further to increased intracellular Ca2+ concentration (Tsien and Tsien, Annu. Rev. Cell Biol. 6:715-760 (1990); Peppelenbosch et al., J. Biol. Chem. 266:19938-19944 (1991)); and (3) activation of Ca2+-dependent K+ channels in the plasma membrane with increased K+ conductance and membrane hyperpolarization (Magni et al., J. Biol. Chem. 261:9321-9327 (1991)). These mitogen-induced early ionic changes, considered critical events in the signal transduction pathways, are powerful therapeutic targets for inhibition of cell proliferation in normal and malignant cells.
One therapeutic approach towards the treatment of diseases characterized by unwanted or abnormal cell proliferation via alteration of the ionic fluxes associated with early mitogenic signals involves the administration of clotrimazole. As discussed above, clotrimazole has been shown to inhibit the Ca2+-activated potassium channel of erythrocytes. In addition, clotrimazole inhibits voltage- and ligand-stimulated Ca2+ influx mechanisms in nucleated cells (Villalobos et al., FASEB J. 6:2742-2747 (1992); Montero et al., Biochem. J. 277:73-79 (1991)) and inhibits cell proliferation both in vitro and in vivo (Benzaquen et al., Nature Medicine 1:534-540 (1995)). Recently, clotrimazole and other imidazole-containing antimycotic agents capable of inhibiting Ca2+-activated potassium channels have been shown to be useful in the treatment of arteriosclerosis (U.S. Pat. No. 5,358,959 to Halperin et al.), as well as other disorders characterized by unwanted or abnormal cell proliferation.
Glaucoma is a disease characterized by increased intraocular pressure. Increased intraocular pressure is associated with many diseases including, but not limited to, primary open-angle glaucoma, normal tension glaucoma, angle-closure glaucoma, acute glaucoma, pigmentary glaucoma, neovascular glaucoma, or trauma related glaucoma, Sturge-Weber syndrome, uveitis, and exfoliation syndrome.
Currently, there are a variety of drugs available that employ different mechanisms to lower intraocular pressure, e.g., timolol, betaxolol, levobunolol, acetazolamide, methazolamide, dichlorphenamide, dorzolamide, brinzolamide, latanoprost, brimonidine, and bimatoprost (see, e.g., U.S. Pat. No. 6,172,054, U.S. Pat. No. 6,172,109, and U.S. Pat. No. 5,652,236). Miotics, beta blockers, alpha-2 agonists, carbonic anhydrase inhibitors, beta adrenergic blockers, prostaglandins and docosanoid are all currently used alone or in combination to treat glaucoma. Miotics and prostaglandins are believed to lower intraocular pressure by increasing drainage of the intraocular fluid, while beta blockers, alpha-2 agonists and carbonic anhydrase are believed to lower intraocular pressure by decreasing production of intraocular fluid thereby reducing the flow of fluid into the eye. All are characterized by side effects ranging from red eye and blurring of vision to decreased blood pressure and breathing difficulties.